Reproduction of the Three-line Genic Male Sterile Line Parent Mian 7MB-1 （Brasscia Napus L.） and Seed Production of F1 Based on Somatic Tissue Culture

[Objective] The aim was to study the reproduction of the three-line genic male sterile （GMS） lineparent Mian7MB-1 （B. NapusL.） and the seed production of F1 through somatic tissue culture. [Methed] Through hybridization, a new breeding material Mian 7MB-1 in three-line genic temporary maintainer line propagated by tissue culture was used to improve the sterile plant rate of rapeseed in dual-purpose recessive GMS line, such as Mian 7AB type, S45AB type, and etc. And then the variety comparative test was performed. [Result] In order to avoid some fertility restoration phenomena occurring during the process of self-reproduction, Mian 7AB was propagated in bulk with somatic tissue culture of temporary maintainer line plant stem. The propagated temporary maintainer line seedlings were applied to the breeding and seed production of net room male sterile line parent, promoting the sterile plant rate of the male sterile line parent to 91.7% -93.5%. The male sterile line parents per hectare were enough for the seed production of hybrid F1 in 7 500 -15 000 hm^2. [ Conclusion ] Compared with the original dual-purpose GMS line, the seed production ultilizing male sterile line with high sterile plant rate greatly reduced the labor, significantly improved the seed yield, ensuring the seed quality and forming a perfect breeding and seed production system.

Study on Seed Germination Conditions and in vitro Tissue Culture of Lilium formolongi

To facilitate the rapid production of lily （Liliumformolongi）, conditions favoring the germination of dormant lily seeds and in vitro regeneration from tissue cultttre were studied, using the germinating plumule as the explants. The optimal inducing conditions for dormant lily dry seeds to germinate were low tempera- ture （ 10 ℃ ） treatment for one hour followed by incubating at 18 ℃ with light illumination. After 8 days of incubation/induction, over 90% of the seeds were ger- minated. MS ＋ 6-BA 1.0 mg,/L ＋ NAA 0.5 mg/L was the most efficient medium formula in lily callus induction. MS ＋ 6-BA 2.0 mg/L ＋ NAA 0.5 mg/L was the optimal medium for calli growth. 1/2MS ＋ NAA 1.0 mg/L was the medium gave the highest root inductive rate.

Tissue Culture of Mini-Papaya

[Objective] The aim was to study the tissue culture of mini-Papaya.[Method] The pretreatment seeds of mini-Papaya were cultured in the MS medium containing 6-BA and IBA of different densities for rapid propagation.[Result] In the condition of aseptic strain,the surface of mini-Papaya peel was uniformly wiped by 75% alcohol,then seeds were removed and washed by aseptic water for 3 times,which was the best sterilization method,and the pollution rate of seed was only 2.52%.After seeds which had been soaked by the equi-volume mix-solution of 1 000 mg/L GA3 and 1 mg/L 6-BA for 18 h were further purified in MS medium,the germination rate of seed,the length of embryo bud and radicle and the height of seedling were 68.42%,2.25,0.80 and 1.52 cm respectively,furthermore the total situation of seedling growth was better.When subculture multiplication medium was MS＋0.5 mg/L 6-BA＋0.1 mg/L IBA medium,proliferation coefficient of subculture multiplication reached the highest （7.92）,and the seedlings grew better.The ratio of vitrified shoots decreasing with the increase of light intensity could reach the lowest level （3.21%） under the light intensity of 3 000 lx.[Conclusion] The research provides reference for studies on tissue culture and rapid propagation of mini-Papaya.

Establishment of Tissue Culture Regeneration System in Bama Hemp (Cannabis sativa L.)

[Objectives]This study was conducted to establish a tissue culture regeneration system in Bama hemp(Cannabis sativa L.).[Methods]Using hemp seeds as explants,a regeneration system was established through explant sterilization,callus induction,callus differentiation,and rooting culture.[Results]The results showed that the best sterilization effect was achieved when sterilizing with 75%ethanol for 30 s,followed by 0.1%HgCl 2 solution for 9 min,with a contamination rate as low as 11.4%.In presence of 3 mg/L 2,4-D and 0.1 mg/L6-BA,the callus induction effect from hemp seeds was better.The formula for better differentiation of callus was MS+2.0 mg/L 6-BA+0.2 mg/L NAA.IBA had a promoting effect on the rooting of hemp aseptic plantlets.The highest rooting rate reached 80%when MS+0.3 mg/L IBA were used.[Conclusions]This study established a hemp seed regeneration system to provide technical support for the conservation and breeding of hemp germplasm resources.

Callus Induction and Plant Regeneration from Mature Seeds of Perennial Ryegrass

Objective The aim was to explore callus induction and plant regeneration of perennial ryegrass, as well as provide the foundation for transgenic research on perennial ryegrass.[ Methed] Mature seeds of perennial ryegrass were used as explants to study the effects of different hormone compositions on callus induction, proliferation and plant differentiation. Result The result showed that the induction rate achieved its highest on 2,4-D of 8 mg/L combining with 6-BA of 0.025 mg/L, which was up to 56.42%. Callus were differentiated after two to three generations, the highest differentiation rate 34.14% was achieved in the medium contained MS medium with 6-BA of 2 mg/L, and the differentiation rate was obviously affected by the callus condition after proliferation. The root inducing medium, containing 0.5 mg/L NAA and MS medium with half of macroelement, gained 98% root inducing rate. Conclusien A high frequency genetic regeneration system was established.

Progress on research of tissue culture of Bletilla striata

Rhizoma Bletillae is in high demand as a traditional Chinese medicine, and the natural resources have been severely damaged due to excessive exploitation. Because Bletilla striata seeds are small and have no endosperm, the seed germination rate is low in natural conditions. Traditional division propagation could not meet the demands of large-scale cultivation. Tissue culture can provide many seedlings in a short time and is more effective and convenient than other methods. Most studies on tissue culture of B. striata selected seeds as explants. This review summarized the processes of the aseptic seed germination pathway. It included such stages as seed germination, proliferation of clusters of buds, induction of root- ing and transplanting of seedlings. Influential factors as well as optimum combination and concentration of the plant growth regulators of each stage were also summarized. The further research on tissue culture of B. striata was also prospected.

Callus induction and plant regeneration from mature bluegrass (Poa pratensis L.) seeds

A protocol was discussed for high efficient plant regeneration from seven bluegrass （Poa pratensis L.） cultivars via an in- direct callus induction and somatic embryogenesis method. Mature seeds were used as explants for callus initiation. Callus induction and proliferation efficiencies were investigated on NB, modified MS （MMS） and MS media, supplemented with 2.0 mg·L^-1 2,4-dichlorophenoxyacetic acid （2,4-D）. The MMS medium performed best. Based on the MMS medium, direct and indirect callus induction effects of bluegrass from mature seeds were compared at the range of 1-5 mg·L^-1 2,4-D contained in the medium. Under the direct callus induction method, the most suitable 2,4-D concentrations varied among cultivars. Under the indirect callus induction method, a significantly high callus induction frequency （93.33%-98.33%） was obtained and there were barely any statistically sig- nificant differences among the tested genetically diverse cultivars. Somatic embryos were promoted on the MMS medium supple- mented with 3 mg·L^-1 2,4-D, 0.1 mg·L^-1 kinetin and 0.8 mg·L^-1 CuSO4. Embryogenetic calli developed into plantlets on the MMS medium containing different concentrations of thidiazuron （TDZ）, and the differentiation frequencies varied in the range from 20.15% to 77.65%. The 0.25 mg·L^-1 TDZ was generally the most suitable concentration for the tested cultivars.

Conversion of Banana Synseed Influenced by the Bead Type and Seed Coat

Banana shoot tips extracted from in vitro grown plant materials were encapsulated in different types of alginate beads. The results obtained after conservation for one month at 4?C indicated that single layered synseeds (where the encapsulated shoot tip was covered by one layer of the artificial endosperm) containing activated charcoal were the best construction in stimulating conversion of synseeds, where they expressed the highest;conversion frequency, number of shoots/synseed, length of shoot and fresh weight/shoot cluster. These parameters decreased when the shoot tips were encapsulated within two layers of endosperm, except the ex vitro conversion. Conversion of banana synseeds was influenced by explant orientation inside the alginate beads, the highest conversion frequency was obtained when the shoot tip was directed upward inside the synthetic endosperm. Synthesis of seed coats around the artificial endosperm improved the conversion frequency, number of shoots/synseed and shoot length.

Sterilization of Seed <i>Carthamus tinctorius</i>(Safflower) Plant and Investigation of the Effectiveness of the Sterilizants

Plant tissue culture studies are one of the pretreatments carried out to increase crop yield by preventing germination in plant seeds. In this research, repeated plant tissue culture studies were conducted with sterilizers specific to safflower seed, which will increase production efficiency but do not cause genetic polymorphism and corrosion in endosperm with 3N chromosomes. Corrosives were used by dilution, and this did not damage the 3N chromosome endosperm, targeting the protein walls of microorganisms on the seed surface without eroding the seed surface, thereby providing biological sterilization. Besides, because it does not contain heavy metals, it did not cause polymorphism, that is, a mutation in the genetic sequence of the seed. Moreover, the environment and the equipment were sterilized with 2 - 3 repetitions, sterilizer treatment, planting, and germination operations were performed in a sterile environment this, in turn, allowed an isolated assessment of the yield of solution G.

伏牛山野生黄精组培快繁体系建立及组培苗质量评估

以伏牛山野生黄精的种子为试验材料,建立其组培快繁体系并对组培苗质量进行评估。结果表明,伏牛山野生黄精种子萌发最佳培养基为1/2 MS+0.5 mg/L ABA,丛生芽诱导最佳培养基为MS+4.0 mg/L 6-BA+0.2 mg/L NAA+0.5 mg/L GA3,丛生芽增殖最佳培养基为MS+3 mg/L 6-BA+0.1 mg/L TDZ+0.5 mg/L 2,4-D+0.4 mg/L NAA,生根最佳培养基为1/2 MS+0.5 mg/L NAA+0.2 mg/L IBA;组培苗分级标准为Ⅰ级苗:株高≥4.2 cm、叶片≥5个,Ⅱ级苗:3 cm<株高<4.2 cm、2<叶片数<5,Ⅲ级苗株高≤3 cm、叶片数≤2个,移栽后成活率为88%。

石橄榄组织培养外植体筛选及培养条件优化

【目的】建立石橄榄组织培养快繁技术体系。【方法】以石橄榄全株为试材,研究外植体的生长情况,筛选出最佳外植体及培养基,优化石橄榄种子萌发、壮苗增殖等培养条件。【结果】石橄榄组织培养最佳外植体为种子,其根、根茎、假鳞茎均未萌发,并出现长霉和化水的情况,而种子均可萌发。石橄榄种子萌发启动最佳培养基配方为1/2MS+6-BA 1.5 mg/L+NAA 0.8 mg/L+6-KT 0.1 mg/L,此条件下石橄榄原球茎变绿色。在基础培养基中分别加入香蕉泥(50 g/L)、土豆泥(50 g/L)、椰汁(50 mL/L)进行壮苗有机物筛选发现,添加香蕉泥的培养基中石橄榄整体长势良好、均一,假鳞茎多,株高与叶长均高于培养基添加土豆泥或椰汁的石橄榄,最终选出50 g/L香蕉泥作为最佳添加有机物加入后续试验的培养基中。石橄榄壮苗优化的最佳培养配方为1/2MS+50 g/L香蕉泥+6-BA 0.5 mg/L+NAA 0.5 mg/L+6-KT 0.3 mg/L,此条件下石橄榄长势好、株高较高。石橄榄壮苗生根的最佳培养基配方为MS+50 g/L香蕉泥+IBA 0.5 mg/L+NAA 0.5 mg/L,此条件下石橄榄假鳞茎多、气生根多立于苗上下。【结论】建立适合石橄榄种子组织培养快繁技术体系,石橄榄种子最佳萌发启动培养基配方为1/2MS+6-BA 1.5 mg/L+NAA 0.8 mg/L+6-KT 0.1 mg/L;最佳壮苗优化培养基配方是1/2MS+50 g/L香蕉泥+6-BA 0.5 mg/L+NAA 0.5 mg/L+6-KT 0.3 mg/L;最佳生根培养基配方是MS+50 g/L香蕉泥+IBA 0.5 mg/L+NAA 0.5 mg/L,为石橄榄的资源保护与利用及其规模化生产奠定基础。

青海茄参种子的萌发特性

【目的】本试验旨在探索青海茄参种子的萌发特性。【方法】以青海茄参(Mandragora chinghaiensis)的种子为试验材料,选择常规播种,低温处理(4℃),组织培养3种方式进行萌发试验。【结果】青海茄参种子经TTC法染色检测,活力为65%;常规播种与低温处理无法使青海茄参种子萌发,只有组织培养的方式获得了种苗;对种子的预处理最适宜的方式为300 mg/L的GA3浸泡16 h;最佳的消毒方式为20%NaClO消毒15 min;最适宜萌发的基本培养基为1/2MS;最有利于青海茄参种子萌发的激素组合为1/2 MS+6-BA 2 mg/L+NAA 0.1 mg/L+GA30.3 mg/L。【结论】探索青海茄参种子的萌发体系,可以缓解青海茄参人工繁殖困难种苗极难获得的瓶颈问题,为进一步研究其药学价值及生物学特性提供新的支持。

尖叶茶藨子种子无菌苗再生体系的建立

以尖叶茶藨子成熟种子为试材,采用不同处理及消毒方式获得无菌苗,再以幼苗茎段为外植体,研究不同植物生长调节剂对丛芽诱导、增殖、壮苗及生根的影响。结果表明:500 mg/L GA_(3)浸泡处理有利于种子的萌发,发芽率可达66.7%,最佳消毒处理为75%乙醇30 s+0.1%HgCl_(2)消毒8 min,污染率为23.3%;最佳丛芽诱导培养基为MS+2.0 mg/L 6-BA+0.05 mg/L NAA,增殖倍数为7.5;最佳壮苗培养基为MS+0.1 mg/L 6-BA+0.05 mg/L NAA+50 g马铃薯泥;最佳生根培养基为1/2MS+2.0 mg/L IBA,生根率为100%,平均生根条数为6.33。炼苗后,移栽到泥炭土∶椰糠∶珍珠岩体积比为3∶1∶1的基质中,成活率达87.5%。该研究建立了尖叶茶藨子种子组培快速繁育技术体系,适用于尖叶茶藨子种苗的规模化生产和应用。

梵净山石斛快速繁殖技术研究

为研究梵净山石斛快速繁殖技术,对其保育提供基础资料,本实验分别以梵净山石斛茎段、种子为外植体进行组培快速繁殖技术研究。结果表明,以茎段为外植体,最佳消毒方法为:75%乙醇消毒30 s,0.1%HgCl_(2)消毒13 min,腋芽诱导最佳组合为:1/2 MS/B_(5)+0.5 mg/L 6-BA+0.2 mg/L NAA,不定芽诱导和增殖最佳组合为:1/2 MS+0.5 mg/L NAA+1 mg/L 6-BA,以种子为外植体,种子萌发和原球茎诱导最佳组合为:MS+1.0 mg/L NAA+1.0 mg/L 6-BA+1 g/L活性炭,原球茎增殖最佳组合为:MS+0.5 mg/L NAA+2 mg/L 6-BA,无菌小苗增殖最佳为:MS+0.5 mg/L NAA+1.0/2.0/3.0 mg/L 6-BA。生根壮苗最佳组合为:MS+0.5 mg/L NAA+1/0.1/0.5 mg/L IBA。

骨组织工程中种子细胞的研究进展

骨缺损是临床常见的疾病,但其治疗困难。在体外将种子细胞进行成骨诱导培养是运用组织工程策略解决骨缺损修复问题的关键因素,其中种子细胞是不可或缺的。目前,常用的种子细胞包括骨髓间充质干细胞、胚胎干细胞、脂肪来源干细胞、脐带间充质干细胞等。近年来,对于种子细胞的研究涉及分离培养鉴定、成骨诱导、信号通路、基因修饰以及联合培养等方面,目的在于揭示成骨机制,提高成骨效率,最终使组织工程技术在临床上得到应用。但种子细胞的培养要求较高,其前沿培养技术主要包括细胞的基因修饰和细胞的共培养。因此,未来需继续寻找或制备容易获取、生物相容性好、成骨能力更强且分化更稳定的种子细胞,这对骨组织工程领域具有重要意义。

大叶藤黄种子的愈伤组织及丛生芽的诱导

大叶藤黄Garcinia xanthochymus种子具有全能萌发的特点,但其生理基础仍然不明确。为了研究其再生生长特性,分别以大叶藤黄成熟和未成熟种子的切片为外植体,MS为基本培养基,探讨添加外源NAA、TDZ、IAA、6-BA、2,4-D等生长调节物质诱导愈伤组织和不定芽的效果。结果表明:(1)愈伤组织的发生主要取决于种子是否成熟。取自成熟种子的外植体,即使不添加外源生长调节物质,诱导率也超过80%;而取自未成熟种子的外植体,仅TDZ(0.25~3 mg·L^(–1))和6-BA(1.5~2 mg·L^(–1))有效,且发生率低于40%。(2)仅6-BA可稳定诱导生成不定芽,最佳方案为取成熟种子外植体在含5.5 mg·L^(–1)6-BA的MS培养基上培养诱导,其发生率可达73%,外植体的平均不定芽发生数量为5.07个。该研究结果进一步证明大叶藤黄种子具有强大的再生生长能力,同时所研究的幼苗再生方案也为大叶藤黄无性繁育提供了可靠途径。

甘薯脱毒及种薯(苗)繁育技术研究

中国是世界上最大的甘薯生产国,然而由于病毒病的存在,严重制约了甘薯产业的发展,建立通用的脱毒组培体系、高灵敏度的检测体系及高效的种薯(苗)繁育体系对甘薯产业发展具有重要意义。对12个主栽品种进行茎尖脱毒处理,建立了通用的脱毒组培体系MS+2 mg/L NAA+1.0 mg/L 6-BA,大多数品种的再生率在58%以上;获得SPFMV、SPCSV、SPLV、SPCFV和SPVG这5种病毒的特异性引物,该引物对病毒检测极限依次为10^(-6)、10^(-1)、10^(-4)、10^(-3)和10^(-3),具有较高的灵敏性;对脱毒品系进行筛选,获得优良脱毒株系甘12和粉薯2-2;建立脱毒种薯(苗)的三级繁育体系,即组培苗(一级)扩繁,温网室基质苗(二级)扦插扩繁和网棚大田苗(三级)繁育生产用种;进行洗苗速率(带根和不带根)对比试验,移栽方式(带根和不带根)与每孔移栽棵数(1~3棵)的双因素试验,结果表明不带根处理可显著提升洗苗速率,育苗前期每孔移栽棵数(1~3棵)在株高、茎粗、节间数方面无显著差异,带根处理前期生长具有优势,但二者成活率无显著差异,并不影响育苗结果。因此,该研究有利于甘薯病毒病防治、脱毒种薯(苗)推广和育苗成本的降低。

一种新的组培大豆种子的消毒方法

为获得一种较好的环境友好型组培大豆种子的消毒方法,本试验通过对三种不同消毒方式下大豆种子组培4 d的萌发数目和污染数目进行统计。研究结果表明:通过不同浓度和不同时间的过氧化氢消毒后,四个品种的萌发数目趋势一致。都是随着15%H_(2)O_(2)消毒时间的延长,种子萌发越多,在灭菌1 h时全部萌发,且达到最大值;随着时间的再次延长,萌发数目减少且受到抑制。浓度为30%H_(2)O_(2)随着灭菌时间的延长,大豆种子萌发抑制程度加深,当灭菌2 h后,将近20%左右的大豆种子受到抑制未萌发。此外用同样的品种进行了其他两种方式的消毒,研究结果发现随着0.1%生汞、5%次氯酸钠消毒时间的增加,污染的种子越来越少,但是萌发数目越来越受抑制,在此试验品种中均没有达到完全消毒的目的。综上所述,选取15%H_(2)O_(2)消毒1 h最为合适,能够保证大豆种子完全萌发且不污染。同时选取国内主栽的15个大豆品种进行试验结果的验证,最终获得了一种新的组培大豆种子的消毒方法。此方法是一种高效、省时、便捷的消毒方式,这为下一步大豆遗传转化试验奠定了基础。

毛报春叶片组培快繁体系建立

为了建立成熟的毛报春组织培养体系,以毛报春(Primula×pubescens)叶片为外植体,采用组培快繁的方法,研究了不同培养基、不同植物生长调节剂种类及浓度配比对愈伤组织诱导和分化、不定芽增殖及生根培养的影响,并对组织培养过程中出现的玻璃化苗现象进行恢复。结果表明:(1)最佳愈伤组织诱导培养基为MS+6.0 g/L琼脂+30.0 g/L蔗糖+1.2 mg/L NAA+1.5 mg/L 6-BA,诱导率可达96.67%,愈伤组织呈绿色,质地紧密。(2)愈伤组织分化最适培养基为MS+6.0 g/L琼脂+30.0 g/L蔗糖+0.5 mg/L NAA+2.0 mg/L 6-BA,分化出的丛生芽生长健壮。(3)最适不定芽增殖培养基为MS+6.0 g/L琼脂+30.0 g/L蔗糖+0.5 mg/L NAA+0.2 mg/L 6-BA,增殖率可达70.33%,苗绿且生长旺盛。(4)最有利于生根诱导的培养基为MS+6.0 g/L琼脂+30.0 g/L蔗糖+0.2 mg/L NAA,生根率可达70.67%,且根粗壮,不易脱落;(5)培养基中添加0.5 g/L活性炭或者3.0 mg/L AgNO 3玻璃化苗恢复效果最好。

喜马拉雅紫茉莉种子消毒及无菌苗培养条件的研究

以剥去外种皮的喜马拉雅紫茉莉种子为外植体,研究不同消毒剂对喜马拉雅紫茉莉种子消毒效果及萌发的影响,并对无菌苗培养基进行筛选。结果表明:(1)种子最适消毒方法为75%乙醇1 min+2%NaClO 15 min,污染率为3.74%,褐化死亡率为1.33%,种子发芽率为94.05%。(2)种子发芽适宜的基本培养基为MS+蔗糖30 g/L+琼脂7 g/L,种子发芽启动时间为4.33 d,发芽率在95%以上。(3)将子叶展开的无菌苗转接到MS+6-BA 0.2 mg/L+蔗糖30 g/L+琼脂7 g/L培养基上,培养至30 d,无菌苗平均叶片数为15.33个,叶片肥厚宽大,茎节间较短,生长旺盛。该研究结果为喜马拉雅紫茉莉无菌苗的培育提供了科学依据和技术支持。